Autotaxin, also known as ATX, ENPP2 or NPP2, short for Ectonucleotide pyrophosphatase phosphodiesterase 2, is an enzyme secreted within the human body. This molecule has been known for generating (LPA) through conversion of lysophosphatidyl-choline (LPC) thereto via lysophospholipase D activity (the removal of choline from the base compound generates LPA). LPA has been realized to contribute to tumor cell growth, unfortunately, as the reactivity within the human body of LPA within certain tissues has resulted, in certain studies, in cancerous growths when present at certain levels. In this manner, then, it has been theorized that the greater the incidence of autotaxin activity within the human body, the greater the possibility of LPA generation. A reduction in the catalytic capabilities of autotaxin to convert the LPC molecule to LPA would theoretically permit an ultimate reduction in possibility of unwanted cell proliferation through reduced LPA presence within a subject's body.
The mechanism of autotaxin in terms of enzymatic activity and catalysis to form LPA resides in its phosphodiesterase capability. LPA can be generated from the cleavage of the phosphodiester bonds of LPC, through the function of a phospholipase enzyme (note Formula I).
In extracellular fluids, this enzymatic catalysis of LPC removes the choline group, leaving LPA, which has a tendency to stimulate cell growth and proliferation as well as chemotaxis. From this, it appears that the motility of tumor cells is increased as well, resulting in properties and gene expression within certain carcinomas (such as, for instance, breast cancer cells), causing further processing into a form that is bioactive and potentially dangerous. Metastasis and oncogenesis of cancer cells appear to occur as well with elevated levels of LPA present within a targeted region. Increased ATX expression has been identified in renal carcinoma, metastatic breast cancer, thyroid carcinoma, Hodgkin lymphoma, and invasive glioblastoma multiforme, as well as other diseases, including multiple sclerosis, obesity, diabetes, Alzheimer's diseases, and chronic pain.
It has thus been determined that the ability to prevent, or at least reduce, the amount of LPA within the human body holds great promise at, likewise, reducing, if not preventing, the onset of certain diseases, most prominently, certain cancers. It has been theorized, as noted above, that autotaxin modifications may prevent the undesirable conversion from LPC to LPA; the ability to actually accomplish such a result has been elusive, however, at least to the degree necessary for effective broad-scale utilization of such a method. Any modification thereof must exhibit an ability to drastically reduce the activity of autotaxin while also, preferably exhibiting oral bioavailability as well.
Autotaxin (ATX, NPP2) was originally identified as an autocrine motility factor in the conditioned media of A2058 melanoma cells. Subsequently, ATX was shown to be, as discussed above, the lysophospholipase D enzyme responsible for synthesis of the bioactive lipid lysophosphatidic acid (LPA) in vivo. Specific, potent inhibitors of ATX are therefore desirable as novel therapeutic leads.
Examples of metal chelators, lipid analogs and non-lipid, small molecules have all been identified as autotaxin inhibitors. Metal chelators such as EDTA, phenanthroline, and L-histidine have been shown to inhibit ATX activity, presumably via interactions with active site divalent metal ions required for function. Lipid analogs represent the largest group of reported ATX inhibitors (see FIG. 1 for structures). LPA and the related bioactive lipid sphingosine 1-phosphate (S1P) were previously shown to function as feedback inhibitors of ATX. This discovery led to the analysis of several LPA and S1P analogs as ATX inhibitors. Reported LPA analogs include fatty alcohol phosphates, Darmstoff analogs, cyclic phosphatidic acid analogs, and phosphonates (VPC8a202, S32826, and JGW-8). One reported S1P analog, FTY720-phosphate, has also been examined as an ATX inhibitor (structures of these types of previous ATX inhibiting compounds are provided below).

While many of these lipid analogs are potent ATX inhibitors, they lack many characteristics seen in 90% of orally bioavailable drugs, and collectively they lack significant structural diversity.
Finally, the third category of reported ATX inhibitors consists of non-lipid, small molecules that collectively extend structural diversity and in general possess physicochemical characteristics more closely related to orally bioavailable drugs. The most efficacious structures previously identified are shown in group Figure A, below. H2L 7905958 (1) was the most efficacious compound from that initial single concentration screen (at 10 μM compound 1 fully inhibited ATX-catalyzed hydrolysis of 1 μM FS-3). Recently, additional small-molecule inhibitors of ATX (group Figures B and C) were identified as potential as metastasis blockers as well. One compound, NSC 48300, in group Figure C, showed essentially 100% inhibition of melanoma metastasis at micromolar concentrations. Although this compound, and the group of compounds similar thereto, exhibited acceptable, if not effective autotaxin inhibition, it is not clear if such compounds are selective to ATX (NPP2).

Furthermore, the previous ATX inhibiting compounds all exhibited certain drawbacks in largescale potential utilization. For instance, past work at ATX inhibition has included, as noted above, L-histidine. Unfortunately, millimolar concentrations were required for any efficacy, and, more importantly, zinc sulfate reversal of this effect (in submillimolar concentrations) suggested an inhibition mechanism involving interaction with the two native active site metal ions thereof. Other potential ATX inhibitors have included the products of ATX-catalyzed hydrolysis of LPC and sphingosyl phosphorylcholine (SPC), LPA, and S1P, respectively. Inhibition of ATX by LPA and S1P suggests that product feedback inhibition may contribute to regulation of ATX function in vivo. Additional reported ATX inhibitors share several common structural features, including a phosphate, thiophosphate, or phosphonate headgroup attached either with or without a linker to an alkyl chain, which can vary in overall length and can be either saturated or unsaturated. However, these compounds lack substantial structural diversity for possible additive or synergistic effect at improving ATX reduction. For oral ingestion purposes, as well, it is of great importance to identify novel non-lipid structural classes capable of inhibiting ATX that are structurally dissimilar from those currently used for this purpose.
It is believed, without relying upon any specific scientific basis, that the lack of diversity in reported ATX inhibitors, as noted above, is due, in part, to the lack of a characterized three-dimensional structure of the enzyme itself. The ATX sequence of over 860 amino acids is divided into several domains, including a central catalytic domain composed of about 400 amino acids. ATX is a member of the nucleotide pyrophosphatase/phosphodiesterase (NPP) family, as well as the alkaline phosphatase superfamily. Crystallographic structures of several alkaline phosphatase superfamily members have been available for decades. These crystal structures show remarkable structural conservation in a small core surrounding the catalytic site, but unfortunately show completely different structural characteristics outside this conserved core. Sequence homology of the alkaline phosphatases with ATX does not exceed 14% and is therefore insufficient for generation of a high quality homology model in any region outside the approximately 100 amino acid structurally conserved core. The recent report of a crystal structure of a bacterial NPP enzyme with 30% identity to the ATX catalytic core domain enabled the development of a structural model of the ATX catalytic domain that may prove useful in structure-based drug design. Although a significant improvement, such a homology model must be applied cautiously as involvement of the c-terminal nuclease-like domain in substrate recognition has been suggested from studies of NPP family domain-swapping chimeras. In any event, these previously reported ATX inhibitors are analogs of LPA, a phospholipid, and are more hydrophobic than is typical of orally bioavailable drugs, thereby creating problems in that area.
As such, there exists a definitive lack in providing effective ATX inhibition (or inactivation) within the current knowledge base in this area, particularly as it concerns compounds that not only exhibit ATX inhibition, but do so selectively for NPP2 alone, as compared with other NPP-type compounds (NPP6 and NPP7, for instance). The determination of proper selective NPP2 inhibiting compounds can thus aid in not only further optimization of autotaxin treatment agents, but also an understanding of the actual amino acid structures outside of the ATX conservative core. As such, although some compounds may promote ATX inhibition as they currently exist, some others with homologous structures (at least to a certain degree) to such effective ATX reduction agents may serve as intermediate compounds for further reaction and/or modification for such an optimization process.
As noted above, previous attempts at such treatments have provided developments of certain classes of compounds that exhibit certain desired results with ATX inhibition. However, the generation of classes that effectively provide increased overall ATX inhibition characteristics has been lacking in the pharmaceutical industry. The present invention provides not only improved ATX inhibiting compounds, but possible intermediates as launching pads into further improvement possibilities within this area as well.